Introduction
Mechanical stresses elicit cellular reactions mediated by chemical signals. Defective
responses to forces underlie human medical disorders
1–4
, such as cardiac failure
5
and pulmonary injury
6
. The actin cytoskeleton’s connectivity enables it to transmit forces rapidly over
large distances
7
, implicating it in these physiological and pathological responses. Here we identify
the actin-binding protein, filamin A (FLNa)
8, 9
as a central mechanotransduction element of the cytoskeleton. We reconstituted a minimal
system consisting of actin filaments, FLNa and two FLNa-binding partners: the cytoplasmic
tail of β-integrin, and FilGAP. Integrins form an essential mechanical linkage between
extracellular and intracellular environments, with β integrin tails connecting to
the actin cytoskeleton by binding directly to filamin
4
. FilGAP is a FLNa-binding GTPase-activating protein specific for Rac, which in vivo
regulates cell spreading and bleb formation
10
. Using Fluorescence Loss After photoConversion (FLAC), a novel high-speed alternative
to FRAP
11
, we demonstrate that both externally-imposed bulk shear and myosin II driven forces
differentially regulate the binding of these partners to FLNa. Consistent with structural
predictions, strain increases β-integrin binding to FLNa, whereas it causes FilGAP
to dissociate from FLNa, providing a direct and specific molecular basis for cellular
mechanotransduction. These results identify the first molecular mechanotransduction
element within the actin cytoskeleton, revealing that mechanical strain of key proteins
regulates the binding of signaling molecules.
Main text
The composite cytoskeleton network in vivo provides dynamic cellular structure and
actively generates movement. A physiological reconstituted in vitro network of actin
and filamin A (FLNa) creates an elastic gel mechanically dominated by the rod-like
actin filaments and crosslinked by flexible FLNa molecules. Applying strain to this
network readily deforms FLNa crosslinks (Fig 1a,b), and the specific structure and
actin binding of FLNa suggest how these deformations might affect FLNa’s interactions
with some of its ~90 other binding partners currently identified
9
.
FLNa is an extended homodimer composed of two identical subunits, each having an N-terminal
actin-binding domain followed by 24 immunoglobulin (Ig) repeats
12
(Fig 1c,d). The actin-binding domains and repeats 1–15 are designated “rod 1”, which
forms a linear structure that binds actin filaments. Repeats 16–23 comprising “rod
2”, however, form compact globular clusters that do not interact with actin filaments
and contain most of FLNa’s binding partner sites. Strain-dependent reversible straightening
of these domains contributes to FLNa-actin network flexibility and may regulate local
binding partner affinity (Fig S1). Here we examine the effects of mechanical strain
on FLNa’s interactions with two key rod 2 binding partners; cytoplasmic β-tail integrin,
which nucleates an extensively characterized signalling
13
and adhesion
14
complex, and FilGAP, a GTPase specific for Rac, a regulator of cellular activity such
as actin assembly
10
. Mechanical strain may regulate partner binding, and we propose that stretching FLNa
crosslinks causes FilGAP to unbind whereas integrin binds more strongly (Figs 1c–d,
S1). Neighboring Ig repeats cover integrin binding sites on FLNa repeats 19 and 21
15, 16
, yet computational simulations suggest that rod 2 of FLNa is highly flexible, and
that physiological forces are sufficient to expose these cryptic sites allowing integrin
to bind
17, 18
(Fig S1a,b). FilGAP binding occurs on each repeat 23, suggesting FilGAP is able to
bind repeat 23 on both subunits simultaneously when unstressed, providing sufficient
avidity to promote FilGAP association with FLNa (Figs 1c, S1c). Mechanical stretching
of FLNa spatially separates repeats 23, preventing FilGAP from binding simultaneously
to both
19
, thus causing it to dissociate (Figs 1d, S1d).
To test these hypotheses and measure the effect of mechanical stress on binding-partner
interactions with FLNa, we reconstituted networks of F-actin and FLNa containing the
binding partner FilGAP or β7-integrin. To quantify the strain-dependent kinetics of
these partners to FLNa, we developed a novel high-speed analogue to FRAP
11
, Fluorescence Loss After photoConversion (FLAC), which takes advantage of the rapid
photo-activation or conversion of photo-activateable fluorescent proteins (PAFPs).
In FLAC, a sample with initially non-fluorescent binding partner is locally pulsed
with a 50 ms 405 nm light, rapidly and permanently activating PAFP-conjugated partner
fluorescence (Figs S4 & S5). Photoactivation fluorescently marks the sample faster
and without the high excitation flux required for conventional photo-bleaching. Post
activation, unbound PAFP rapidly diffuses away, decreasing the fluorescent signal,
while bound PAFP dissociates more slowly. The time-dependent decay of PAFP intensity
reveals the kinetics of the FLNa binding partner, as a slower decay curve indicates
slower unbinding, providing a direct high-speed assay of dissociation.
We tested the utility of these PAFP constructs in assaying binding kinetics by reconstituting
F-actin, PAFP-labeled binding partners, with different forms of FLNa that have higher
or lower affinity for β7 integrin or FilGAP. Consistent with immunoprecipitation data
(Fig S3b,c), the fluorescence decay of PA-GFP β7 integrin was faster in wild-type
FLNa networks, than in the del41 mutant (movie S1), demonstrating relatively stronger
binding in the del41 mutant compared to wild-type. The fluorescence decay of PA-GFP
FilGAP was slower in wild-type FLNa networks, than in the M2474E mutant (movie S2),
also in agreement with immunoprecipitation data (Fig S3a).
We measured the mechanosensitive aspect of PAFP-binding partner interactions with
FLNa. We sheared networks of F-actin and FLNa containing PAFP tagged FilGAP or β7-integrin
in a precise and highly controlled fashion using a microscope stage comprised of a
stationary coverslip for the bottom of the sample and a piezo-controlled linear actuator
at the top. When the FLNa-F-actin network was not strained, β7-integrin had a characteristic
exponential decay time of 0.4 +/− 0.1s. The application of a shear strain, γ=0.28,
increased this time to 1.0 +/− 0.1s (Fig 2a). The change in fluorescence decay rate
describes how the geometric state of FLNa affects dissociation of β7 -integrin; thus,
mechanically stretching FLNa molecules enhanced the β7 -integrin binding. In contrast,
FilGAP behaved qualitatively oppositely: unstrained networks had a characteristic
fluorescence decay time of 2.3 +/− 0.4s, which decreased to 0.3 +/− 0.1 s when a 0.28
shear strain was applied (Fig 2b). FLNa does not permanently cross-link actin, and
by unbinding and rebinding on the time-scale of ~6 min (Fig S6), it dynamically allows
the network to relax to an unstressed state. After 10 min under strain the network
had sufficient time to dissipate internal stress through FLNa remodeling, and the
fluorescence decay time increased to 3.4 +/− 0.5 s, demonstrating the reversibility
of strain modulated FilGAP binding to FLNa (Fig 2b).
While the application of unidirectional shear revealed the effects of strain on partner
binding to FLNa, cells commonly generate internal stresses using molecular motors
such as myosin. To examine the effects of cytoskeleton-induced stress, and as a physiological
complementary technique to external shear, we included myosin II in the networks to
generate contractile stress
20
(Fig S9 and movie S3). We allowed the composite network to assemble and come to an
unstressed equilibrium state over ~6 hours after the incorporated myosin II had ceased
contracting by enzymatically exhausting the pool of added ATP, and dynamic FLNa remodeling
had dissipated internal stresses. For unstressed FLNa, we measured β7 -integrin and
FilGAP fluorescence decay times of 1.6 +/− 0.1 s and 1.5 +/− 0.1 s, respectively (Fig
3a,c). Including photolabile ‘caged’ ATP in the sample allowed us to release fresh
ATP and restart myosin motor activity
21, 22
, which contracts the actin network and strains FLNa crosslinks. Myosin stressed FLNa
increased the integrin unbinding time to 2.5 +/− 0.2 s, while decreasing the FilGAP
unbinding time to 0.9 +/− 0.1 s (Fig 3a,c). The application of either external shear
or myosin contraction resulted in increased integrin binding and decreased FilGAP
binding, demonstrating the robust yet opposite behaviors of these FLNa binding partners.
The FLNa crosslinked actin cytoskeleton is a large percolated network that readily
transmits mechanical signals over long intra-cellular distances due to the filamentous
actin structure, yet FLNa is mechanosensitive at nanometer molecular deformations.
This is in contrast to focal adhesion mechanosensitivity, which detects local mechanics
and is limited to small spatial and strain scales due to their size and connectivity
23, 24
.
In conclusion, we have developed in vitro systems to determine quantitative protein-protein
interactions under mechanical force. Using PAFPs with the FLAC technique provides
the advances in time-resolution necessary for measuring transient kinetics, without
the harsh intensity or duration of bleaching exposure required for FRAP. The results
presented here establish FLNa as the first mechanotransductive substrate within the
cytoskeleton, and highlight the utility of in vitro systems combined with the power
of the FLAC technique to determine quantitative responses of specific proteins.
Mechanotransduction in vitro provides the biological specificity necessary for understanding
how these complex regulatory signals may operate in vivo. Cellular mechanotransduction
has been shown to induce rapid biochemical activity over long distances
25
. Since mechanical stimuli induces relatively large local deformations that decrease
in magnitude with distance from application site, FLNa mechanotransduction in vivo
likely provides a rapid, distance-sensitive biphasic response by binding or unbinding
integrins or FilGAP, respectively, due to the transmitted strain. Physiologically,
the localization and binding of these proteins determine their activity. Strain induced
binding of integrin to FLNa may compete with talin binding to integrin
26
, thus providing a mechanosensitive switch for integrin activation and adhesion. FLNa’s
homodimer structure may induce clustering of integrin, thereby reinforcing adhesion
and concentrating signaling molecules at a specific location. FilGAP, when unbound
from FLNa, relocates to the plasma membrane where it inactivates Rac
10
. Active Rac levels profoundly impact cell movement
27
and increased Rac activity in FLNa deficient cells correlates with increased apoptosis
28
. Moreover, our measurements are consistent with in vivo studies demonstrating that
Rac activity and expression appear to be force-regulated by FilGAP-FLNa interactions,
since inhibiting FLNa or FilGAP increases Rac levels, yet applying local forces to
wild-type cells causes FilGAP to decrease Rac expression
28
. Since FLNa does not change FilGAP’s catalytic activity, mechanically-induced redistribution
alone might explain its regulation in vivo. Force-dependent conformational changes
in structure required for mechanical-regulation have been observed in many proteins,
including FLNa in vivo
29, 30
. By identifying FLNa as the first mechanosensitive element within the cytoskeleton,
we have clarified how Rac and integrin activity may be regulated by a specific molecular
mechanotransduction pathway. Identifying mechanotransduction elements may direct unique
therapeutic approaches by correcting or modulating mechanosensitive binding.
Methods summary
PAFP fluorophore synthesis
PAFP fluorophore cDNA was inserted into binding partners, creating PAFP labeled s7
integrin and FilGAP. Solubility and correct binding of labeled partners was confirmed
using western blots (Fig S3).
FLAC methodology
An external 405 nm laser was coupled into a Leica SP5 confocal microscope and used
to illuminate a central ~2μm spot for 50 ms, converting the PAFP from its dark to
fluorescent state (Figs S4 & S5). The decay in fluorescence intensity, I(t), of the
activated fluorophores was monitored and fit with the exponential:
I
(
t
)
=
a
∗
e
(
−
t
/
k
)
+
c
where k is the time constant of characteristic dissociation. Given k values represent
best fits +/− 95% confidence intervals.
Sample cell composition
Shear cell samples consisted of 24 μM actin, 0.12 μM FLNa, 1xFB, 2μM Alexa 546 phalloidin,
and either PAGFP FilGAP or β7 integrin, and were sheared in a piezo-driven shear cell
(see Supplemental information). Sheared FLAC measurements for strained networks were
acquired approximately 5–10 s after shear. Myosin samples included 24 μM actin, 0.12
μM FLNa, 1μM myosin II, 1xAB, 2 μM caged ATP, and 2μM Alexa 546 phalloidin, and PAGFP
FilGAP, or 2μM Alexa 488 phalloidin and PA-mCherry β7 integrin. Samples were allowed
to polymerize and consume available ATP over 6 hours. FLAC measurements were then
performed on the ATP-free unstressed network. Subsequently, the caged ATP (Sigma)
was released by a 4 s exposure to a diffuse 50 mW 365 nm LED light (Prizmatix Israel),
and within 3 s the network could be seen to homogenize under myosin contraction (Fig
S9 and movie S3). FLAC measurements were then repeated in this active myosin stressed
network to quantify the strain dependent binding activity.
Supplementary Material
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